Carbon dioxide shapes parasite-host interactions in a human-infective nematode

Skin-penetrating nematodes infect nearly one billion people worldwide. The developmentally arrested infective larvae (iL3s) seek out hosts, invade hosts via skin penetration, and resume development inside the host in a process called activation. Activated infective larvae (iL3as) traverse the host body, ending up as parasitic adults in the small intestine. Skin-penetrating nematodes respond to many chemosensory cues, but how chemosensation contributes to host seeking, intra-host development, and intra-host navigation – three crucial steps of the parasite-host interaction – remains poorly understood. Here, we investigate the role of carbon dioxide (CO2) in promoting parasite-host interactions in the human-infective threadworm Strongyloides stercoralis. We show that S. stercoralis exhibits life-stage-specific preferences for CO2: iL3s are repelled, non-infective larvae and adults are neutral, and iL3as are attracted. CO2 repulsion in iL3s may prime them for host seeking by stimulating dispersal from host feces, while CO2 attraction in iL3as may direct worms toward high-CO2 areas of the body such as the lungs and intestine. We also identify sensory neurons that detect CO2; these neurons are depolarized by CO2 in iL3s and iL3as. In addition, we demonstrate that the receptor guanylate cyclase Ss-GCY-9 is expressed specifically in CO2-sensing neurons and is required for CO2-evoked behavior. Ss-GCY-9 also promotes activation, indicating that a single receptor can mediate both behavioral and physiological responses to CO2. Our results illuminate chemosensory mechanisms that shape the interaction between parasitic nematodes and their human hosts and may aid in the design of novel anthelmintics that target the CO2-sensing pathway.

In vitro activation assay Single-worm CO 2 chemotaxis assay Strategy for CRISPR/Cas9-mediated disruption of the Ss-gcy-9 gene using homology-directed repair.HA = homology arm.Successful integration of the repair template will insert mRFPmars into the Ss-gcy-9 gene, thereby disrupting the gene.Expression of the mRFPmars reporter is driven by the promoter for the Ss-act-2 gene [6].The wild-type locus surrounding the Ss-gcy-9 target site is amplified by the F1 x R1 primer set.Successful integration of the 5' HA of the repair template is confirmed by the F1 x R2 primer set.Primer binding sites shown are approximate.D. Gel showing representative genotyping of a wild-type and Ss-gcy-9 -/-iL3 (left), and the corresponding genotyping scheme (right).iL3s with a homozygous disruption of the Ss-gcy-9 gene and successful integration of the repair template will lack the wild-type (wt) band that amplifies a 926 bp region around the CRISPR target site and will have a 5' integration band (5') that amplifies a 934 bp region spanning the end of the 5' homology arm and part of the upstream genomic DNA.The control (ctrl) band amplifies a portion of the Ss-act-2 gene and serves to confirm that genomic DNA is present [6].The binding sites for the genotyping primers are shown in C. E. Schematic of a single-worm CO 2 chemotaxis assay.Left, side view of the assay plate; right, top view of the assay plate.A single iL3 is placed in the center of a 9 cm agar plate and a CO 2 gradient is established by pumping 40% CO 2 into one side of the plate and air into the other side through holes in the plate lid.At the end of the 10-min assay, the side of the plate containing the iL3 is determined.iL3s that remain in the center zone of the plate ("neutral," right) are not counted.F. Schematic of an in vitro activation assay [7][8][9].iL3 are incubated in host-like conditions (DMEM, 37°C, 5% CO 2 ) for 21 h, after which fluorescent dye is added to the wells.Activated iL3s that resume feeding ingest the dye, resulting in pharyngeal fluorescence.Non-activated iL3s that have not resumed feeding do not have a fluorescent pharynx.Schematic is from Gang et al., 2020 [7].  is not the result of exposure to host-like conditions; rather, it is specific to iL3as.iL3s that were exposed to in vitro activation conditions but did not activate ("non-activated iL3s") were repelled by CO 2 , while animals that activated under the same conditions were attracted to CO 2 .Graph shows responses to 40% CO 2 .Each data point represents a single CO 2 chemotaxis assay.Lines in violin plot indicate medians and dotted lines indicate interquartile ranges.****p<0.0001,Mann-Whitney test.n = 10-12 trials per condition.C. Schematic of the chamber used to track the locomotion of iL3s and iL3as in response to acute CO 2 pulses.Animals are placed on an agar surface and covered by the gas-delivery chamber.Their locomotion is then video-recorded during gas exposure through the transparent 6 cm arena.Schematic is from Banerjee et al., 2023 [10] and was adapted from Rojo Romanos et al., 2018 [11].D. The stimulus delivery paradigm for tracking responses to acute CO 2 pulses.In control assays, animals were exposed to 30 s of air, followed by 30 s of air from a second air tank, followed by 30 s of air from the original air tank.In experimental assays, animals were exposed to 30 s of air, followed by 30 s of 2.5% CO 2 , followed by 30 s of air.E. Mean smoothed instantaneous speeds (± SEM) of animals from air control assays for iL3s (left) and iL3as (right).Responses of each life stage during the middle 30 s air pulse are quantified in Figure 4. n = 16-22 animals per condition.F. Representative time-series images of iL3s illustrating the different types of CO 2 -evoked turns.Movements defined as turns were either reverse-coupled omega turns, where the head and tail made an angle of less than 30° (left column, t = 1 sec); reverse-coupled omega turns, where the head and tail touched (middle column, t = 2 sec); and reverse-coupled delta turns (right column, t = 1 sec) [12].To perform calcium imaging from iL3as, an in vitro activation assay was first performed with iL3s expressing a gcy-9p::strYC3.60transgene.iL3as were identified based on the presence of Alexa Fluor NHS Ester 594 in the pharynx (A); the red Alexa Fluor dye was used instead of FITC so that the dye would not be visible during calcium imaging.iL3s that were exposed to the same activation assay conditions but did not activate ("non-activated iL3s") were identified by the lack of Alexa Fluor dye in the pharynx Primer for testing for genomic integration of the 5' homology arm Table S3.Summary of microinjections introducing the CRISPR constructs for targeted disruption of the Ss-gcy-9 gene.Table columns show, from left to right, the number of freeliving adults injected (P0), the number of F1 iL3 screened, the number of transgenic iL3s collected, the number of transgenic iL3s genotyped, the number and percentage of genotyped iL3s with integration of the repair template, and the number and percentage of genotyped iL3s with a homozygous disruption of Ss-gcy-9.Percentages in the last two columns were calculated based on the number of red iL3s genotyped by PCR.The bottom row displays the totals from all experiments.

Figure S2 .
Figure S2.Disruption of the Ss-gcy-9 gene using CRISPR/Cas9-mediated targeted mutagenesis.Related to Figure 2. A. Intron-exon diagram of the gcy-9 genes of C. elegans and S. stercoralis.The Ss-gcy-9 gene structure is based on the gene annotations from WormBase ParaSite [4, 5].White bars indicate untranslated regions (UTRs); lines indicate introns.Scale bar = 100 bp.B. The predicted domains of the C. elegans and S. stercoralis GCY-9 proteins.The CRISPR target site is in the predicted guanylate cyclase domain of the S. stercoralis GCY-9 protein.Domain predictions are from InterPro and the figure was generated using ProSite.C.Strategy for CRISPR/Cas9-mediated disruption of the Ss-gcy-9 gene using homology-directed repair.HA = homology arm.Successful integration of the repair template will insert mRFPmars into the Ss-gcy-9 gene, thereby disrupting the gene.Expression of the mRFPmars reporter is driven by the promoter for the Ss-act-2 gene[6].The wild-type locus surrounding the Ss-gcy-9 target site is amplified by the F1 x R1 primer set.Successful integration of the 5' HA of the repair template is confirmed by the F1 x R2 primer set.Primer binding sites shown are approximate.D. Gel showing representative genotyping of a wild-type and Ss-gcy-9 -/-iL3 (left), and the corresponding genotyping scheme (right).iL3s with a homozygous disruption of the Ss-gcy-9 gene and successful integration of the repair template will lack the wild-type (wt) band that amplifies a 926 bp region around the CRISPR target site and will have a 5' integration band (5') that amplifies a 934 bp region spanning the end of the 5' homology arm and part of the upstream genomic DNA.The control (ctrl) band amplifies a portion of the Ss-act-2 gene and serves to confirm that genomic DNA is present[6].The binding sites for the genotyping primers are shown in C. E. Schematic of a single-worm CO 2 chemotaxis assay.Left, side view of the assay plate; right, top view of the assay plate.A single iL3 is placed in the center of a 9 cm agar plate and a CO 2 gradient is established by pumping 40% CO 2 into one side of the plate and air into the other side through holes in the plate lid.At the end of the 10-min assay, the side of the plate containing the iL3 is determined.iL3s that remain in the center zone of the plate ("neutral," right) are not counted.F. Schematic of an in vitro activation assay[7][8][9].iL3 are incubated in host-like conditions (DMEM, 37°C, 5% CO 2 ) for 21 h, after which fluorescent dye is added to the wells.Activated iL3s that resume feeding ingest the dye, resulting in pharyngeal fluorescence.Non-activated iL3s that have not resumed feeding do not have a fluorescent pharynx.Schematic is from Gang et al., 2020[7].

E
Figure S3.BAG neurons respond to CO 2 .Related to Figure3. A. Ss-BAG neurons do not respond to an air control.Graph shows the calcium response (mean ± SEM) of the Ss-BAG neurons to a 30 s pulse of air.Calcium response was measured using the ratiometric calcium indicator yellow cameleon YC3.60.Beige square shows the timing and duration of the air pulse.n = 15 iL3s.B. Heatmap of the Ss-BAG calcium responses.Each row shows the response of a single animal.Response magnitudes (% ΔR/R 0 ) are color-coded according to the scale shown to the right.Rows are ordered by hierarchical cluster analysis.Black bar shows the timing and duration of the air pulse.C. Schematic of the construct used for cell-specific silencing of the Ss-BAG neurons.The Sr-gcy-9 promoter was used to drive specific expression of tetanus toxin (TeTx) in the Ss-BAG neurons.The self-cleaving peptide P2A was used to express strTeTx and strmScarlet-I from the same promoter so that animals expressing TeTx could be identified based on expression of mScarlet.D. The TeTx transgene is expressed specifically in the Ss-BAG neurons.Left, representative DIC/epifluorescence overlay image of an S. stercoralis iL3 expressing TeTx in the Ss-BAG neurons.The head region indicated by the white box is enlarged to the right.Right, enlarged epifluorescence image of a BAG neuron; head is to the left.E. The synthesized cDNA sequence of the Strongyloides-codon-optimized tetanus toxin gene (strTeTx) that was used to silence the Ss-BAG neurons of S. stercoralis iL3s.The stop codon was removed for co-transcription with strmScarlet-I in the bicistronic vector shown in C. Purple font indicates a synthetic intron sequence.
Figure S4.iL3as are attracted to CO 2 .Related to Figure4. A. iL3as are attracted to CO 2 concentrations as high as 40% in a CO 2 chemotaxis assay.Assays were conducted at 37°C to mimic intra-host temperature conditions.****p<0.0001relative to the 0% CO 2 control, two-way ANOVA with Dunnett's post-test.n = 12-16 trials per condition.Graph shows medians and interquartile ranges.B. CO 2 attraction is not the result of exposure to host-like conditions; rather, it is specific to iL3as.iL3s that were exposed to in vitro activation conditions but did not activate ("non-activated iL3s") were repelled by CO 2 , while animals that activated under the same conditions were attracted to CO 2 .Graph shows responses to 40% CO 2 .Each data point represents a single CO 2 chemotaxis assay.Lines in violin plot indicate medians and dotted lines indicate interquartile ranges.****p<0.0001,Mann-Whitney test.n = 10-12 trials per condition.C. Schematic of the chamber used to track the locomotion of iL3s and iL3as in response to acute CO 2 pulses.Animals are placed on an agar surface and covered by the gas-delivery chamber.Their locomotion is then video-recorded during gas exposure through the transparent 6 cm arena.Schematic is from Banerjee et al., 2023[10] and was adapted from Rojo Romanos et al., 2018[11].D. The stimulus delivery paradigm for tracking responses to acute CO 2 pulses.In control assays, animals were exposed to 30 s of air, followed by 30 s of air from a second air tank, followed by 30 s of air from the original air tank.In experimental assays, animals were exposed to 30 s of air, followed by 30 s of 2.5% CO 2 , followed by 30 s of air.E. Mean smoothed instantaneous speeds (± SEM) of animals from air control assays for iL3s (left) and iL3as (right).Responses of each life stage during the middle 30 s air pulse are quantified in Figure4.n = 16-22 animals per condition.F. Representative time-series images of iL3s illustrating the different types of CO 2 -evoked turns.Movements defined as turns were either reverse-coupled omega turns, where the head and tail made an angle of less than 30° (left column, t = 1 sec); reverse-coupled omega turns, where the head and tail touched (middle column, t = 2 sec); and reverse-coupled delta turns (right column, t = 1 sec)[12].

Figure S5. iL3as show an enhanced CO 2 -
Figure S5.iL3as show an enhanced CO 2 -evoked calcium response in Ss-BAG neurons compared to non-activated iL3s.Related to Figure5.A-B.To perform calcium imaging from iL3as, an in vitro activation assay was first performed with iL3s expressing a gcy-9p::strYC3.60transgene.iL3as were identified based on the presence of Alexa Fluor NHS Ester 594 in the pharynx (A); the red Alexa Fluor dye was used instead of FITC so that the dye would not be visible during calcium imaging.iL3s that were exposed to the same activation assay conditions but did not activate ("non-activated iL3s") were identified by the lack of Alexa Fluor dye in the pharynx (B).The red fluorescence in the posterior part of the animal in B is due to intestinal autofluorescence.C-D.iL3as (C) show a larger CO 2 -evoked calcium response in the Ss-BAG neurons than non-activated iL3s (D), as confirmed by statistical comparison of the maximum responses to the CO 2 stimulus (E). *p<0.05,Mann-Whitney test.n = 28-29 animals per life stage.
Figure S5.iL3as show an enhanced CO 2 -evoked calcium response in Ss-BAG neurons compared to non-activated iL3s.Related to Figure5.A-B.To perform calcium imaging from iL3as, an in vitro activation assay was first performed with iL3s expressing a gcy-9p::strYC3.60transgene.iL3as were identified based on the presence of Alexa Fluor NHS Ester 594 in the pharynx (A); the red Alexa Fluor dye was used instead of FITC so that the dye would not be visible during calcium imaging.iL3s that were exposed to the same activation assay conditions but did not activate ("non-activated iL3s") were identified by the lack of Alexa Fluor dye in the pharynx (B).The red fluorescence in the posterior part of the animal in B is due to intestinal autofluorescence.C-D.iL3as (C) show a larger CO 2 -evoked calcium response in the Ss-BAG neurons than non-activated iL3s (D), as confirmed by statistical comparison of the maximum responses to the CO 2 stimulus (E). *p<0.05,Mann-Whitney test.n = 28-29 animals per life stage.